PROJECTSUMMARY/ABSTRACT: Proper cortical network development and function is reliant on the generation, maturation, and activity of numerouscelltypesinadditiontotheircomplexcell-cellinteractions.Essentialtothisprocessistheoutputof glutamatergicpyramidalneurons(PyNs),whichishighlymodulatedbyinhibitoryGABAergicinterneurons.One subset of interneurons that exerts powerful control over PyN spiking is the chandelier cell (ChC),which forms connections specifically at the site of action potential initiation in PyNs, referred to as the axon initial segment (AIS).DuetotheuniqueconnectionsformedbetweentheterminalsofChCaxonalarborsandtheAISsoflarge populations of spiking PyNs, ChCs are physiologically poised to regulate the output of excitatory cortical networks. As a result, it is not surprising that disruptions in ChC biology have been linked to autism spectrum disorder(ASD)andschizophrenia,debilitatingmentalhealthdisordersresultingfromaberrantneuronalnetwork activity.DespitetheimportanceofChCs,virtuallynothingisknownaboutthemolecularfactorsgoverningtheir selective innervation at the AIS of neocortical PyNs. By performing a novel, in vivo RNA interference (RNAi) screenagainstPyNAIS-specificand-enrichedadhesionmolecules,thisstudyintriguinglyrevealedanessential rolefortheaxonalcelladhesionmoleculeL1CAMinChC/PyNAISinnervation.Specifically,L1CAMknockdown inneocorticalPyNswasfoundtosignificantlyreducePyNAISinnervationbyChCs,thusidentifyingL1CAMas the only known molecule to date to regulate ChC/PyN subcellular target recognition. This application aims to elucidate how L1CAM governs ChC/PyN AIS innervation and to identify the presynaptic binding partner(s) of L1CAM on ChC axon terminals. To this end, Aim 1 will use molecular tools to disrupt interactions between L1CAMandtheAIScytoskeletontodeterminewhethercytoskeleton-mediatedL1CAMclusteringisnecessary forproperChC/PyNAISinnervation.Inaddition,molecularreplacementstrategieswillbeutilizedtoinvestigate L1CAM?ssubcellulardistributionontheAISandaxonofneocorticalPyNsinvivo.Finally,Aim1willdetermine whetherPyNL1CAMisrequiredearlyonfortheestablishmentand/orlateronforthemaintenanceofChC/PyN innervation using RNAi technology in combination with in utero electroporation (IUE)- and adeno-associated viral-basedstrategies.Aim2focusesonidentifyingthepresynapticbindingpartner(s)ofL1CAMonChCaxon terminals.BasedonmutantL1CAMscreening,neuropilin-1(Nrp1)ishypothesizedtoserveasthepresynaptic partnerofL1CAMonChCsnecessaryforproperChC/PyNinnervation.Totestthis,experimentsutilizingChC- targetingIUE,RNAitechnology,andconditionalNrp1knockoutmicewillbeemployedtodepleteNrp1inChCs. Molecularandbiochemicalstrategieswillalsobepursuedtodefinethecriticaldomain(s)ofNrp1necessaryfor L1CAMbindingandproperChC/PyNsubcellulartargetrecognition.Together,theproposedstudieswillprovide vital information on the mechanisms governing ChC/PyN AIS innervation and, as such, shed new light on the connectivitydefectsunderlyingdebilitatingmentaldisorders.